Reduced toxicity in alcoholic beverages

ABSTRACT

The present invention relates to reduced toxicity of functional alcoholic beverage composition comprising distilled alcohol, deionized water, 18β-Glycyrrhizin or 18α-Glycyrrhizin and a sugar alcohol or sugars, having pH in the range of 4.0-9.0. More particularly, alcoholic beverage composition comprises distilled alcohol, deionized water, 18β-Glycyrrhizin or 18α-Glycyrrhizin and a sugar alcohol/sugars as hepato-protectants. The present invention provides an alcoholic beverage for reducing hepatotoxicity caused by its consumption and a process to manufacture the said alcoholic beverage.

The following specification particularly describes the invention and themanner in which it is to be performed.

FIELD OF INVENTION

The present disclosure provides an alcoholic beverage having reducedhepato-toxicity. The invention also relates to a process for thepreparation of the said beverage.

BACKGROUND OF THE INVENTION

Ethanol consumption could lead to 60 medical conditions. Acute as wellas chronic toxic effect of ethanol may ensue in irreversible organdamage (Das S. K. et. al., Indian journal of Biochemistry & Biophysics,2010, Vol. 47, 32). The widely accepted forms of alcoholic liverdiseases (ALD) are simple fatty liver (steatosis), which is reversiblewith abstinence, fatty liver accompanied by inflammation(steato-hepatitis) leads to scar tissue formation (fibrosis), thedestruction of the normal liver structure (liver cirrhosis), which mayor may not improve with abstinence and subsequently lead to liver cancer(hepatocellular carcinoma). In 2010, WHO suggests 10% of the adultpopulation in the United States suffering from alcohol use disorders andliver cirrhosis is the 12^(th) leading cause of death in United States(Alcohol and Health, Focus on: Alcohol and the Liver, 2010, Vol. 33, No.1 and 2, 87). It is known that 5% of the ethyl alcohol i.e. ethanol(hereinafter alcohol), ingested by a human being is excreted unchangedwhile the remaining 95% is degraded to acetaldehyde. Alcohol is rapidlyabsorbed from the GI tract. In fasting state the peak blood alcoholconcentration reaches within 30 minutes. Distribution is rapid withtissue levels approximating blood concentrations. Liver accounts fornearly 90% of alcohol metabolism the remainder is excreted through thelungs & urine. The typical adult can metabolize 7-10 g of alcohol/hour(U.S. Pat. No. 7,666,909B2).

The primary pathway of alcohol metabolism, when consumed in low tomoderate amount, is mainly catalyzed in the cytoplasm of hepatocytes byalcohol dehydrogenase (ADH) to form acetaldehyde. The accumulation ofNADH (excess reducing equivalents) in the liver plays a role in liverdamage seen more prominently with chronic alcohol use. Acetaldehydeproduced through microsomal ethanol oxidation system (MEOS) initiallyrepresents a minor pathway of ethanol oxidation probably accounting forless than 10%, of the liver capacity to oxidize ethanol.

At higher alcohol level (>100 mg/dl), MEOS is dependent on CYP450 (2E1,1A2 & 3A4) plays significant role in alcohol metabolism using NADPH as acofactor & O₂. Catalase is especially capable of oxidizing ethanol infasting state in the presence of hydrogen peroxide generating system.Acetaldehyde is oxidized in the liver via mitochondrial nicotinamideadenine dinucleotide (NAD⁺) dependent aldehyde dehydrogenase (ALDH) toacetate. Activity of ALDH is nearly 3 times lower that ADH, henceaccumulation of Acetaldehyde takes place. Acetate is further metabolizedto acetyl CoA and can enter min TCA cycle or synthesis fatty acids. Eachof these pathway results in the formation of free radicals (likereactive oxygen species {ROS}) with concomitant changes in the cellsredox state (i.e. in the ratio of NADH to NAD⁺ results in production ofmore NADH (Nicotinamide Adenine Dinucleotide (NAD⁺) reduced by twoelectrons). The cell has a limited capacity to oxidize NADH back to NAD⁺in mitochondrial respiratory chain at the maximum capacity of thissystem, which determines the kinetics of the reaction. The redox statein relation to alcohol metabolism causes inhibition of NAD⁺-mediatedenzyme reactions typical to the normal metabolism of the hepatocyte. Thecitric acid cycle is affected the most as it gets inhibited. This leadsto positive NADH/NAD ratio, which is considered the most importantreason for the development of alcohol-induced fatty liver. The maximumcapacity of the mitochondrial respiratory chain depends on the overalllevel of metabolism of the body. The consequence of altered redox stateincludes Hypoxia (oxygen deficit cell). The other plausible pathway ofalcohol induced hepatotoxicity includes excess production ofpro-inflammatory cytokines by gut-endotoxin stimulated Kupffer cells.ROS is mainly generated in association with the mitochondrial electrontransport system; it is also produced by CYP2E1 and by activated Kupffercells in the liver. Both acute and chronic alcohol consumption canincrease ROS production, which leads to oxidative stress through avariety of pathways mentioned above [(Zakhari, S. Alcohol Research &Health, 2006, 29, 4, 245), (Wheeler M. D. et al, Free Radical Biology &Medicine, 2001, Vol. 31, No. 12, 1544), (Koop, D. R., Alcohol Research &Health, 2006, 29, 4, 274), (U.S. Pat. No. 7,666,909B2)].

The mechanisms involved by which alcohol causes cell injury are complexand combination of several inter-related pathways. ROS react primarilywith the cell membrane (tight junction becomes more permeable) and inturn leaks lipopolysaccharides (LPS), as a consequence impaired gutstructural integrity. The increases in transaminase enzymes [aspartateamino-transferase (AST) and alanine aminotransferase (ALT)] indicatecellular leakage and loss of functional integrity of cell membrane (Yueet. J, 2006). Loss of cellular integrity affects hepato-biliary functionleading to elevated alkaline phosphatase (ALKP) activities withconcurrent increase in serum bilirubin level and decrease in the totalplasma protein content. Both increases and decreases in the levels ofROS can lead to apoptosis of hepatocytes (Wheeler M. D. Alcohol Res.Health, 2003; 27, 300). For the cell to function normally, GSH iscritical to protect itself against ROS generated during activity of themitochondrial respiratory chain. Alcohol consumption rapidly depletesGSH levels; alcohol interferes with Cytochrome c to leak from themitochondria into the cytosol, which can activate enzymes known ascaspases that can trigger apoptosis.

ROS induces LPO [ROS reacting with Malondialdehyde (MDA), 4-hydroxynonenal (HNE)] and recognized as important starting place of hepatocytesdamage. Endotoxin-activated Kupffer cells affects mitochondria leadingto release of ROS (hydrogen peroxide radical, hydroxyl radical,particularly superoxide radical) and several cytokines (viz., Tumournecrotic factor {TNF-α}) leading to hepatocytes necrosis and apoptosis.It has been established by clinical studies that patients with alcoholicliver disease have increased levels of the inflammatory cytokines IL-1,IL-6, and TNF-α as well as the chemokine IL-8 and other cytokines.

Alcohol might enhance the sensitivity of hepatocytes, consequently whichcould lead to an increased production of ROS in the mitochondria. ROScould activate a regulatory protein called nuclear factor kappa B(NFκB), which plays critical role in regulation of immune response andcontrols the activities of numerous genes, including those thatexpresses TNF-α & its receptor as well as genes encoding proteins thatpromote apoptosis. Thus, a vicious cycle would be established in thehepatocytes: TNF-α promotes ROS production, which in turn activatesNFκB, leading to enhanced production of additional TNF-α and itsreceptor as well as to production of factors that promote apoptosis.This cycle eventually alters the structure of the hepatocytes, impairstheir function, and can lead to hepatocyte apoptosis. TNF-α alsofacilitates hepatocyte regeneration by promoting the proliferation[(Wheeler M. D. Alcohol Res Health, 2003; 27, 300), (Molina P., Happel,K. I., Zhang P., Kolls J. K., Nelson S., Focus on: alcohol and theimmune system. Alcohol Res. Health, 2010, 33 (1 & 2), 97)1)].

TGF-β (transforming growth factor beta) might be involved in thedevelopment of alcohol-induced liver damage, which could cause thehepatocytes to produce molecules like trans-glutaminase, cytokeratinsthat are normally responsible for giving the cells their shapes. Inexcess, these molecules are cross-linked to form microscopic structurescalled Mallory bodies, which are markers of alcoholic hepatitis. TGF-βcan also contribute to liver damage by activating stellate cells. In anormal state, these cells primarily serve to store fat and vitamin A inthe liver. When activated, stellate cells produce collagen, the majorcomponent of scar tissue it leads to the development of liver fibrosis.Alcohol might trigger the activation of TGF-β and thereby contribute tothe initiation of apoptosis if this molecule enters the blood in higherconcentrations (Wheeler M. D., Alcohol Res. Health, 2003; 27, 300).

Acetaldehyde or ROS with DNA or protein or protein building blocks andROS with MDA or MAA (mixed MDA-acetaldehyde-protein adduct) or HNE etc.in the cell could form stable or unstable adduct, which could becarcinogenic, immunogenic, induce inflammatory process, damage to themitochondria etc. [(Zakhari, S. Alcohol Research & Health, 2006, 29(4)245); (D. Wu, Alcohol Research & Health, 2106, 27, 4, 277); (WheelerM. D., Alcohol Res. Health, 2003; 27, 300); (Molina P., Happel K. I.,Zhang P., Kolls J. K., Nelson S., Focus on: alcohol and the immunesystem; (Alcohol Res. Health, 2010, 33, Vol. 1 & 2, 97); (Neuman M. G.,Cytokine-central factor in alcoholic liver disease, Alcohol Res. Health,2003, 27, 307)].

Varieties of endogenous enzymatic and non-enzymatic mechanisms haveevolved to protect cells against ROS. This includes the superoxidedismutases (SOD), which remove O₂ ⁻; Catalase (CAT) and the glutathioneperoxidase (GP_(X)) system, which remove H₂O₂ and non-enzymaticlow-molecular-weight antioxidants such as reduced glutathione (GSH),Vitamin E, Vitamin C, Vitamin A, Ubiquinone, Uric acid, and bilirubin.But these are capable to protect the cells to limited extent. Additionalprotection could be achieved by orally administrating the glutathioneprecursor like S-adenosyl-L-methionine (SAMe), N-acetyl cysteine (NAC)or anti-oxidant like Vitamin E. Vitamin C, plant bioactives (gallicacid, quercetinete) etc. (D. Wu, Alcohol Research & Health, 2006, 27, 4,277).

PRIOR ART OF THE INVENTION

Literature discloses alcoholic beverages with various types ofadditives. The following literature exists in the field of thisinvention and has been considered in entirety.

US Patent Publication No. 20100086666 discloses alcoholic beverages inwhich a protein like casein hydrolysate to enhance smoother taste andgives some nutritional benefit to the consumer.

Das S. K. et. al. (Indian Journal of Biochemistry & Biophysics, 2010,vol 47, 32) describes concomitant treatment of resveratrol or vitamin Ewith alcohol in mice ameliorates; alcohol induced oxidative stress,angiogenesis process and aid in controlling immune-modulatory activity.

US Patent Publication No. 20100086666 discloses alcoholic beverages,which comprises phenol like epigallocatechingallate (EGCG),epigallocatechine (EGC), epicatechin (EC), epicatechingallate (ECG),proanthocyanin, tannin and quercetin etc. known to reduce oxidativestress by scavenging free radicals generated by alcohol.

US Patent Publication No. 7666909B2 reveals alcoholic beveragescomprising D-Glyceric acid and its salts enhancing the metabolism ofalcohol reducing the adverse event caused due to alcohol consumption.

GA or Matrine (Mat) alkaloid isolated from S. flavescens alone, orGA+Mat, when administered to rat models of hepatic fibrosis induced byabdomen injection of dimethyl nitrosamine (DMN) in acetaminophenoverdosed mice, reduces the mortality by attenuatingacetaminophen-induced hepatotoxicity. This is probably due to reducednumber and area of γ-GT positive foci. In addition, GA+Mat had aprotective effect on immunosuppression, a strong non-specificanti-inflammatory effect, and an effect of reducing the incidence ofsodium and water retention (W. Xu-yingae, Chemico-BiologicalInteractions, 181 (2009) 15-19).

WO No. 2008/055348A1 discloses that alcoholic beverages comprisingturmeric reduces hangover.

Das S. K. et al. (Indian Journal of Experimental Biology, 2006, Vol 44,791) reveals concomitant treatment of lecithin with Vitamin B complex orVitamin E with alcohol in Wistar rats was performed. It was establishedthat lecithin with Vitamin B complex with alcohol was promisingtherapeutic approach than Vitamin E with alcohol in allaying oxidativestress.

El-Fazaa S. et al. (Alcoholism & Alcoholism, 2006, Vol. 41, No 3, 236)exemplifies alcoholic beverages comprising resveratrol inhibits thealcohol induced lipid peroxidation and have protective effect againstinjury.

WO 1989004165A1 or EP0336960A4 divulges alcoholic beverages withcombination of any one or more sugars from the group consisting ofD-Galactose, D-Lactose, D-Xylose. L-Fructose, D-Mannitol, D-Sorbitol,D-Glucose etc.

JP 06014746 discloses alcoholic beverages comprising a glycoside ofquercetin, divalent metallic ion and licorice extract (Glycyrrhizin).This beverage enhances alcohol metabolism and hashepatopathy-suppressive activity, due to ethanol and acetaldehyde. Thus,it reduces hangover.

CP Patent Publication No. 1736270 discloses liver-protecting drinkconstituting Chitosan oligosaccharide, glycyrrhizin, aqueous extract ofkudzuvine flower and aqueous extract of hovenine.

US Patent Publication No. 20090196951 reveals alcoholic beveragescomprising resveratrol a strong anti-oxidant, also activates the Sirtuin1 (SIRT1) and Peroxisome proliferator-activated (PPAR)-gammacoactivator-1[PGC-1′] gene, which are key regulator of energy andmetabolic homeostasis.

JP2008266203 and EP0502554 discloses an increase in amount of an enzymeactivity of the Reactive oxygen species (ROS) scavenging enzyme groupsuch as superoxide dismutase, catalase or peroxidase with one or morekinds of substances selected from the group consisting of Erythritol,Mannitol, Sorbitol and Xylitol.

CN1448497 discloses an alcoholic drink comprising of ethanol andGlycyrrhizin, but a synergistic mixture of alcohol withhepato-protectants that include certain sugar alcohols or sugars asintegral part of the present composition, apart from Glycyrrhizin hasnot been described.

CN101744865 discloses a method of producing a liver protecting tabletcomprising Xylitol and Glycyrrhizin. CN101744865 focuses on a method forpreparing Xylitol liver tablets and nowhere demonstrates biologicalactivity of such tablets. Moreover, the present patent is focused to analcoholic beverage having reduced toxicity and a method of preparing thesame. The present application demonstrates a synergistic mixture ofalcohol with hepato-protectants that include certain sugar alcohols orsugars as integral part of the composition and such synergistic mixtureoffers a good degree of hepato-protection.

Various other prior art documents are known (US 20080226787, U.S. Pat.No. 3,282,706, U.S. Pat. No. 1,720,329, U.S. Pat. No. 4,537,763, U.S.Pat. No. 8,524,785) where glycyrrhizin and sugar alcohols like Mannitol,Erythritol, Xylitol etc. have been used for imparting various functionsin the beverages as non-nutritive sweetening agent having low calorificvalue or as flavoring agent, but the aspect of hepato-protection has notbeen disclosed.

Documents are available in prior art, which show that Glycyrrhizin,sugar alcohols and sugars are independently known to exhibithepato-protective activity, but their combination to exhibit synergistichepato-protection has not been reported so far. Applicant in thisapplication reports for the first time synergistic activity imparted bya combination of 18β or α-Glycyrrhizin and sugar alcohols, moreparticularly 18β/α-Glycyrrhizin and D-Mannitol exhibiting exemplifiedsynergistic hepato-protection to provide a beverage with reducedtoxicity.

SUMMARY OF THE INVENTION

The present disclosure relates to an alcoholic beverage, particularly toalcoholic distilled spirits like vodka, flavored vodka, whisky, etc.having reduced hepato-toxicity comprising distilled alcohol, deionizedwater, glycyrrhizin and a sugar alcohol or sugar having a pH in therange of 4.0-9.0.

More particularly the invention provides an alcoholic beverage havingreduced hepatotoxicity comprising distilled alcohol, deionized water,18β-Glycyrrhizin or 18α-Glycyrrhizin and a sugar alcohol or sugar. Theinvention also relates to a process for the preparation of the saidbeverage. The exemplified reduced hepato-toxicity provided by thebeverage has been achieved by synergistic hepato-protection exhibited bythe combination of 18β or 18α-glycyrrhizin and a sugar alcohol/sugarpresent in the said alcoholic beverage.

OBJECTS OF THE INVENTION

An object of the present invention is to provide an alcoholic beveragehaving reduced toxicity.

Another object of the present invention is to provide an alcoholicbeverage having synergistic activity and providing enhancedhepato-protection.

Yet another object of the present invention is to provide a beveragecomprising hepato-protective agent(s) to achieve the reducedhepato-toxicity.

Yet another object of the present invention is to provide an alcoholicbeverage comprising 18β-Glycyrrhizin or 18α-Glycyrrhizin to achieve thereduced hepato-toxicity.

Yet another object of the present invention is to provide an alcoholicbeverage comprising hepato-protective agent(s) like sugar alcohols andsugar.

Yet another object of the present invention is to provide an alcoholicbeverage comprising the sugar alcohols selected from D-Mannitol,D-Erythritol, D-Xylitol and like.

Yet another object of the present invention is to provide an alcoholicbeverage comprising sugars selected from D-Xylose, D-Mannose, D-Sucroseand D-Lactose.

Still another object of the present invention is to provide an alcoholicbeverage comprising pH adjusting agent(s), flavoring agent(s).

Further object of the present invention is provide an alcoholic beveragecomprising optionally of the flavoring agents selected from vanilla,strawberry and like.

Still another object of the present invention is to provide an alcoholicbeverage having acceptable taste, flavor, odor, clarity and buzz factor.

Another important object of the present invention is to provide aprocess for the preparation of alcoholic beverage composition comprising(a) alcohol or alcohol:water mixture (b)18β-Glycyrrhizin/18α-Glycyrrhizin (c) sugar alcohol or sugar (d) pHadjusting agents and optionally a flavoring agent.

Still another object of the present invention provides an alcoholicbeverage composition having enhanced hepato-protection.

The alcoholic beverage is for use in a method of amelioration ofdiseases involving acute and chronic alcoholic toxicity like alcoholicliver diseases (ALD) like steatosis.

BRIEF DESCRIPTION OF THE TABLES

-   Table 1: % Protection of D-Mannitol-   Table 2: % Protection of D-Xylitol & D-Erythritol-   Table 3: % Comparative Protection of 18β and 18α-Glycyrrhizin-   Table 4: % Protection and % Synergism of 18β-Glycyrrhizin-Mannitol    combinations-   Table 5: Comparative % Protection and % Synergism of 18β or    18α-Glycyrrhizin-Mannitol combinations-   Table 6: Comparative % Protection and % Synergism of    18β-Glycyrrhizin-Mannitol, Xylitol & Erythritol)-   Table 7: Comparative data of % Protection and % Synergism of (180    Glycyrrhizin/Mannitol, Xylitol & Erythritol)-   Table 8: % Protection of Sucrose, Mannose, Xylose & Lactose-   Table 9: % Protection and % Synergism of (18β-GA: Sucrose, Mannose,    Xylose & Lactose)

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention provides a beverage, morespecifically an alcoholic beverage having reduced hepato-toxicitycomprising distilled alcohol, deionized water, 18β or 18α-Glycyrrhizinand a sugar alcohol or sugar and having pH in the range of 4.0-9.0. Moreparticularly the hepato-toxicity is caused by the intake of alcohol. Thereduced hepatotoxicity of the beverage of the present invention isachieved by the enhanced hepato-protective activity provided by thesynergistic combination of 18β or 18α-Glycyrrhizin and a sugar alcoholor Glycyrrhizin and a sugar incorporated in the said alcoholic beverage.The synergistic effect of the components has been established by dosedependent study for hepato-protection of 18β or 18α-Glycyrrhizin, sugaralcohol and a combination of Glycyrrhizin and sugar alcohol/sugar byperforming experiment on animal models.

Ingredient Description:

Glycyrrhizin (or Glycyrrhizic acid or Glycyrrhizinic acid: abbreviatedas GA) is the chief sweet-tasting constituent of Glycyrrhiza glabra(liquorice) root. It has also been given intravenously in Japan as atreatment for hepatitis C and as an emulsifier and gel-forming agent infoodstuff and cosmetics. Glycyrrhizin (GA) is a triterpenoid saponinglycoside. II is available as in racemic or pure form of 2 isomers:18β-Glycyrrhizin and 18α-Glycyrrhizin. Hepato-protective mechanism of GAis due to its aglycone, glycyrrhetic acid, which inhibits both freeradical generation as well as lipid peroxidation. 18α-GA has anti-hepatofibrosis effect—it is frequently used as a hepato-protective agent. Thesweetness of GA has a slower onset than sugar, and lingers in the mouthfor some time. GA is partly absorbed as an intact drug. (W. Xuyinga et.al.) Chemico-Biological Interactions 181 (2009) 15-19), (T, Zing et. al.Chinese Journal of Modern Applied Pharmacy 2006, 02, 15-19). GA and itsmetabolites exhibit steroid-like anti-inflammatory activity, due, inpart, to inhibition of Phospholipase A2 activity, an enzyme critical tonumerous inflammatory processes. They inhibit hepatic metabolism ofaldosterone and suppress hepatic 5-α-reductase. Because Cortisol andaldosterone bind with the same affinity to the mineralocorticoidreceptor, an increase in renal Cortisol will result in ahyper-mineralocorticoid effect (Akamatsu, H. Planta Med., 1991, 57:119-121), (Armanini, D., Clin. Endocrinol. 1983, 19: 609).

GA completely suppressed viral antigen expression possibly by causing adecrease in the negative charge on the cell surface and/or by decreasingthe membrane fluidity thereby preventing Hepatitis A virus entry incells by receptor mediated endocytosis (W. Xu-Yinga et. al.,Chemico-Biological Interactions 181 (2009) 15-19).

GA induces phase II enzymes involved in the detoxification and excretionof carcinogenic or toxic substances and other antioxidant enzymesresponsible for maintaining a balanced state between freeradicals/oxidants and the antioxidants within the cellular environment.Oxidative injury in AR mice (Aldose reducrase deficient mice) is reducedby GA, by increasing GSH content and decreased MDA formation in a dosedependent manner. Concomitant decreases were observed in glutathioneperoxidase (GPx), catalase (CAT), total antioxidant capacity (TAOC) andSOD activities in AR mice. IFN-α, or type II interferon, is a cytokinethat is critical for innate and adaptive immunity against viral andintracellular bacterial infections and for tumour control. GA led to asignificant, increase of IFN-α level in medicine treated mice. IL-4 is acytokine that induces differentiation of naive helper T cells (Th0cells) to Th2 cells. Upon activation by IL-4, Th2 cells subsequentlyproduce additional IL-4 (Xiao-Lan Li Int. J. Mol. Sci. 2011, 12, 905).GA could increase infection resistance as [monocyte chemo-attractant(chemotactic) protein-1] is a CC chemokine MCP-1 inhibitor (UnitedStates Patent Application 20060116337).

The mice were treated intra-peritoneally with CCl₄ (0.5 ml/kg). Theyreceived GA (50, 300, 200, 400 mg/kg) 24 h and 0.5 h before and 4 hafter administering CCl₄, This protection is likely due to the inductionof heme oxygenase-1 and the down-regulation of pro-inflammatorymediators (Biol Pharm Bull. 2007, 30, 10, 11898). 18α-GA coulddose-dependently inhibits CCl₄ induced liver fibrosis, by promoting theproliferation of hepatocytes, but inhibited that of Hepatic stellatecells (HSCs) GA blocks the translocation of NF-kB into the nucleus; thiscould suppress the activation and induce the apoptosis of HSCs (Q Ying,Med Sci. Monit., 2012, 18, 1: BR24).

GA was shown to attenuate histological hepatic changes and significantlyreduced serum levels of AST, ALT, and lactic dehydrogenase (LDH), at allthe indicated times. GA also significantly inhibited hepatocyteapoptosis by down-regulating the expression of caspase-3 and inhibitingthe release of Cytochrome c from mitochondria into the cytoplasm. Theanti-inflammatory activity of GA may rely on the inhibition of releaseof tumour necrosis factor-α, myeloperoxidase activity, and translocationof nuclear factor-kappa B into the nuclei. GA also up-regulated theexpression of proliferating cell nuclear antigen, implying that it mightbe able to promote regeneration of livers harmed by LPS. In summary, GAmay represent a potent drug protecting the liver againstendotoxin-induced injury, especially after massive hepatectomy(Brazilian journal of Medical and Biological Research, 2007, 40, 1637).Pretreatment with GA (50 mg/kg) and the MMP inhibitor (5 mg/kg)suppressed increases in serum levels of ALT and AST in mice treated withLPS/Gal N due to a down-regulation of MMP-9 (J Pharm Pharmacol. 2008,60, 1, 91).

The metabolic syndrome (MetS) is a cluster of metabolic abnormalitiescomprising visceral obesity, dyslipidaemia and insulin resistance (IR).Oral administration of 50 mg/kg of GA for one week could counteract thedevelopment of visceral obesity and improve dyslipidaemia via selectiveinduction of tissue lipoprotein lipase (LPL), expression and a positiveshift in serum lipid parameters respectively, and retard the developmentof IR associated with tissue steatosis (Lipids Health Dis. 2009, 29, 8,31).

Diammoniumglycyrrhizinate (DG) protected mice against Concanavalin A(ConA)-induced elevation of serum ALT levels and apoptosis ofhepatocytes; DG may possibly protect the liver from injury via twopathways: direct protection of hepatocytes from apoptosis through anIL-6 dependent way and indirect inhibition of T-cell-mediatedinflammation through an IL-1 independent way (Int Immunopharmacol. 2007October: 7(10): 1292).

Magnesium isoglycyrrhizinate 100 or 150 mg once daily, drugs areeffective and safe treatment for chronic liver diseases (Zhoiighua GanZang Bing Za Zhi. 2009, 11, 847).

A sugar alcohol is a kind of alcohol prepared from sugars. These organiccompounds are a class of polyols, also called polyhydric alcohol,polyalcohol, or glycitol. They are white, water-soluble solids thatoccur naturally and are used widely in the food industry as thickenersand sweeteners. Sugar alcohols such as Mannitol, Erythritol, Sorbitol,Xylitol etc., which are chemically stable can be used as a radicalscavenger (hydroxyl radical). Similarly, it has been found thatcompounds like Erythritol, Mannitol, Sorbitol, Xylitol etc. up-regulateddifferent types of superoxide dismutase (SOD) like Cu/Zn-, Mn- andEC-SOD isozymes. In particular, the SOD activity of the erythritol-addedgroup increased by 2-5 times. Further it is reported that diabetics havea low SOD activity due to the Maillard reaction, because the Maillardreaction remarkably causes a decrease in the SOD activity (US PatentApplication 20100037353 A1). Mannitol containing hyperosmolar solutionhas been shown to protect ethanol-induced gastric mucosal damage(Gharzouli K, Exp. Toxic. Pathol., 2001; 53: 175). Both rats and humansabsorb and metabolize partially the Mannitol ingested in gastrointestinal tract (GIT). However, intestinal microflora convert Mannitolin to more absorbable form. In rat, absorbed mannitol is converted in tohepatic glycogen probably via fructose (J. Nutr. 1985, 115: 890). Themechanism of protecting living cells by Mannitol is not fullyunderstood.

The beverage comprises of certain other ingredients like pH adjustingagent(s), and flavoring agent(s) etc.

Some important embodiments of the beverage of the present invention areas follows:

An important embodiment of the present invention relates to a beveragehaving reduced toxicity.

Yet another embodiment of the present invention relates to an alcoholicbeverage having reduced hepato-toxicity.

Yet another embodiment of the present invention relates to an alcoholicbeverage comprising hepato-protective agent(s) to achieve the reducedhepato-toxicity.

In an important embodiment of the present invention, the beveragecomprises of 18β-Glycyrrhizin in combination with sugar alcoholsselected from the group consisting D-Mannitol, D-Xylitol, D-Erythritoland mixtures thereof and reducing or non-reducing sugars selected fromD-Xylose, D-Mannose, D-Sucrose and D-Lactose and mixtures thereof.

In yet another important embodiment of the present invention, thebeverage comprises of 18α-Glycyrrhizin in combination with sugaralcohols selected from the group consisting D-Mannitol, D-Xylitol,D-Erythritol and mixtures thereof.

In an important embodiment, the beverage composition comprises18β-Glycyrrhizin in the range of 0.05 to 0.4%, preferably 0.1 to 0.3%and D-Mannitol, D-Xylitol, D-Erythritol, D-Xylose, D-Mannose, D-Sucrose,D-Lactose and mixture thereof is in the range of 0.5 to 3.0%, preferably1.0 to 2.5%.

In an important embodiment, the beverage composition comprises18β-Glycyrrhizin in range of 0.05 to 0.3%, preferably 0.1 to 0.3% andD-Mannitol, D-Xylitol, D-Erythitol and mixtures thereof is in the rangeof 0.5 to 3.0%, preferably 1.0 to 2.5%.

In an important embodiment, the most preferable combination ofhepato-protective agents is a combination of 18β-Glycyrrhizin or18α-Glycyrrhizin and D-Mannitol.

In an important embodiment, the beverage composition comprises18β-Glycyrrhizin in the range of 0.05 to 0.3% and the D-Mannitol is inthe range of 0.5 to 3.0% and preferably 18β-Glycyrrhizin in the range of0.1 to 0.3% and the D-Mannitol is in the range of 1.0 to 2.5%.

In an important embodiment, the beverage composition comprises18α-Glycyrrhizin in the range of 0.1 to 0.3% and the D-Mannitol in therange of 1.0 to 2.5%.

In yet another embodiment, the process for the preparation of alcoholicbeverage composition comprising steps of (a) obtaining alcohol or wateror a mixture thereof, (b) mixing 18β-Glycyrrhizin or 18α-Glycyrrhizinwith the alcohol or water or a mixture of alcohol and water of step (a),(c) adding sugar alcohol or sugar to the mixture of step (b), (d)adjusting the pH of the resulting solution of step (c) between 4.0-9.0,(e) optionally adding the flavoring agent and (t) obtaining the requiredalcoholic beverage composition.

Still another embodiment of the present invention is to provide analcoholic beverage composition comprising the pH adjusting agent(s).

In yet another embodiment, the pH adjusting agent is an organic orinorganic base/buffer, preferably selected from potassium sorbate orsodium phosphate (monobasic or dibasic or tribasic).

Further embodiment of the present invention provides a beverageoptionally comprising of flavoring agents selected from, vanilla andstrawberry.

Still another embodiment of the present invention is to provide abeverage having acceptable taste, flavor, odor, clarity and buzz factor.

In a further embodiment of the present invention variation in dosages ofsugar alcohols, glycyrrhizin and a combination of sugar alcohols and 18βor 18α-Glycyrrhizin has also been evaluated for its hepato-protectiveactivity.

The scope of the present invention also includes the study in respect ofacute and chronic hepatotoxicity caused by the variation in the alcoholdosage and its time of duration in administration.

Still another embodiment of the beverage composition relates toproviding reduced hepato-toxicity.

Yet another embodiment of the beverage composition is the use in amethod of amelioration of diseases involving acute and chronic toxicitysuch as alcoholic liver diseases (ALD) like steatosis, steatohepatitis,fibrosis, liver cirrhosis and hepatocellular carcinoma etc. which arecaused by alcohol induced toxicity.

Another important embodiment of the present invention is that thebeverage composition can be packed as ready-to-drink produce in foodgrade bottles, cans, tetra packs, pouches, etc. The packaging can bedone by conventional methods.

For the establishment of synergism existing in the formulation of thepresent invention, markers/marker enzymes viz. SOD, Catalase, GPx, TNF-αwere primarily taken into consideration for evaluating the % synergism.However, enzymes ALT, AST, ALKP and MDA were also analyzed to supportthe same.

Reasons for Estimating ALT, AST, ALKP:

Chronic misuse of alcohol changes marker enzymes of liver functions suchas serum aspartate aminotransferase and alanine aminotransferase (AST,ALT), alkaline phosphatase (ALKP) and so these enzymes were studied.ALT and AST are found in hepatocytes but AST is also found in skeletaland myocardial cells. In alcohol related liver damage, the AST iselevated more than the ALT, at least in part as a reflection of alcoholrelated skeletal damage. This is the reverse of the normal pattern inacute hepatocellular disease (for example acute viral hepatitis) wherethe ALT exceeds the AST.ALKP is an enzyme in the cells lining the biliary ducts of the liver.ALKP levels in plasma will rise almost concomitantly with liver diseaserelated with altered bile production and/or secretion and chronic liverdiseases.

Reasons for Estimating Oxidative Stress Markers (MDA, AntioxidantEnzymes [SOD, CAT, Glutathione Peroxidase Etc.] Reduced Glutathione[GSH]):

Alcohol metabolism in the liver results in the formation reactive oxygenspecies (ROS). Alcohol also stimulates the activity of cytochrome P450,which contribute to ROS production. Further, alcohol can alter thelevels of certain metals in the body, thereby facilitating ROSproduction. Finally, alcohol reduces the levels of agents that caneliminate ROS (i.e., endogenous antioxidants). The resulting state ofthe cell, known as oxidative stress, can lead to cell injury. ROSproduction and oxidative stress in liver cells play a central role inthe development of alcoholic liver disease.MDA (Malondialdehyde) is the end product of cell membrane lipidperoxidation. ROS degrade (oxidative degradation) polyunsaturated fattyacids of cell membrane resulting cell damage. The extent of lipidperoxidation can be well correlated with tissue MDA content.SOD (Superoxide dismutase) catalyzes the breakdown of the superoxideradical into oxygen and hydrogen peroxide. Liver cells are enriched withSOD as it is the major organ related with metabolism numeroussubstances.CAT (Catalase) catalyzes the conversion of hydrogen peroxide (H₂O₂) towater and oxygen. This enzyme is localized to peroxisomes in mosteukaryotic cells.GPx (Glutathione peroxidase) is the most abundantly available in thecytoplasm of most of the cells. It neutralizes hydrogen peroxide (H₂O₂)in presence of GSH.

(GSH-reduced glutathione, GSSG-oxidized glutathione)GSH is the most abundant antioxidant in aerobic cells. GSH is criticalfor protecting the cells from oxidative stress, acting as a free radicalscavenger and inhibitor of lipid peroxidation. (GSH also participates inthe degradation of H₂O₂ by glutathione peroxidases (GPx). The ratio ofreduced glutathione (GSH) to oxidized glutathione (GSSG) is an indicatorof cellular health (status of cellular redox potential). In normalhealthy conditions GSH constituting nearly 90% of cellular glutathione(i.e., GSH/GSSG is around 9). However, the GSH/GSSG ratio is reduced inROS related disorders.

Reasons for Estimating Tumor Necrotic Factor Alpha (TNF-α):

Alcohol consumption increases the translocation of endotoxins fromintestine to portal circulation and interacts with Kuppfer cells(immunocytes) leading to secretion of several pro-inflammatory cytokinesincluding tumor necrotic factor alpha (TNF-α).

Based on the Above Description, we Identified Some Key Marker andJustify the Importance of the Parameter Chosen:

SOD, Catalase & GPx: In system SOD catalyzes the dismutation ofsuperoxide to H₂O₂. GPx and Catalase then independently convert thisH₂O₂ to water. SOD together with GPx and catalase form the main enzymedefense against harmful effect of ROS.GSH is the main endogenous antioxidant that protects cells fromxenobiotics including alcohol. Alcohol is known to deplete GSH levels onthe process to neutralize oxidants. Apart from this, endogenousglutathione-glutathione peroxidase system acts as an importantantioxidants and cyto-protective machinery in the hepatocytes exposed toethanol. Thus, depletion of cellular GSH level plays an important rolein ethanol-mediated hepato-cellular dysfunction.The following tables (1 to 9) illustrate the % of hepato-protection ofindividual ingredients, combination of ingredients and the % synergismexhibited using respective combinations. All animal experiments wereconducted for a period of one month by per oral administration of 2.5g/kg dose of alcohol.

TABLE 1 % Protection of D-Mannitol Sample GSH SOD etc. TNF-α ALT etc MDACode Man % % Prot. % Prot. % Prot. % Prot. % Prot. A 0.5 10.35 12.717.19 12.26 19.17  3 1 20.06 19.32 16.74 20.37 31.63 B 1.5 25.76 26.2129.89 25.94 48.56 C 2.5 31.53 35.83 31.46 29.71 50.8 11 3 32.37 36.0830.76 29.48 50.31

TABLE 2 % Protection of D-Xylitot & D-Erythritol GSH SOD etc TNF-α ALTetc MDA % Prot. % Prot. % Prot. % Prot. % Prot. Xyl %   1% 19.76 18.9115.77 17.62 26.9 2.5% 35.57 36.88 30.05 26.72 45.38 Ery %   1% 18.7117.94 16.57 17.84 24.71 2.5% 37.29 36.29 35.96 32.13 48.61

TABLE 3 % Comparative Protection of 18β and 1.8α-Glycyrrhizin Sample GSHSOD etc TNF-α ALT etc MDA Code GA % % Prot. % Prot. % Prot. % Prot.Prot. % 18β-GA D 0.1 3.29 11.45 7.64 8.38 15.97 U 0.2 12.1 16.72 12.3113.25 27.12 W 0.3 19.1 27.95 21.18 20.99 46.35 X 0.4 31.34 31.05 29.2826.42 56.74 18α-GA 4 0.1 8.93 14.33 10.58 11.98 15.1 5 0.3 16.96 25.8423.45 18.3 41.69

TABLE 4 % Protection and % Synergism of 18β-Glycyrrhizin-Mannitolcombinations Sample GA Man GSH GSH SOD etc SOD etc TNF-α TNF-α ALT etc.ALT etc. MDA MDA Code % % % Prot. % Syn. % Prot. % Syn % Prot. % Syn %Prot. % Syn % Prot. % Syn H 0.1 2.5 48.24 38.51 60.15 26.65 50.56 29.3140.35 10.52 85.62 28.23 L 1 2.5 83.29 10.45 78.75 21.31 87.64 29.9952.35 −11.15 93.29 −20.87 O 0.3 2.5 61.95 22.43 71.57 13.44 69.63 32.2849.4 −1.09 76.54 −21.21 M 0.4 2.5 76.38 21.55 79.83 20.59 81.62 34.3853.15 −4.17 80.41 −25.23 C 0.1 0.5 17.64 28.76 25.34 3.72 19.16 29.2 217.32 39.63 12.78 4 0.1 1 29.58 26.68 39.33 28.1 32.68 34.04 29.13 5.2555.41 16.41 12 0.1 3 45.53 27.68 58.15 22.74 47.2 22.92 37.23 0.37 70.876.93

TABLE 5 Comparative % Protection and % Synergism of 18β or18α-Glycyrrhizin - Mannitol combinations Sample GSH GSH SOD etc SOD etcTNF-α TNF-α ALT etc ALT etc MDA MDA Code Man % % Prot. Syn % % Prot. %Syn % Prot. % Syn % Prot. % Syn % Prot. Syn % 18β-GA % 4 0.1 1 29.5826.68 39.33 28.1 32.68 34.04 29.13 5.25 55.41 16.41 H 0.1 2.5 48.2438.51 60.15 26.65 50.56 29.31 40.35 10.52 85.62 28.23 O 0.3 2.5 61.9522.43 71.57 13.44 69.63 32.28 49.4 −1.09 76.54 −21.21 1.8α-GA % 6 0.1 132.74 12.94 42.42 26.01 34.05 24.63 30.97 −0.29 54.16 15.9 8 0.1 2.552.68 30.2 60.16 19.8 53.21 26.57 41.35 3.51 76.6 16.24 10 0.3 2.5 57.4418.46 69.06 12.57 68.1 24.02 46.49 −1.35 75.8 −18.05

TABLE 6 Comparative % Protection and % Synergism of 18β-Glycyrrhizin-Mannitol, Xylitol & Erythritol) SOD SOD GSH GSH etc. % etc.% % % TNF-α TNF-α Prot. Syn Prot. Syn % Prot. % Syn 0.10%   1% GA % Man% 39.33 28.1 29.58 26.68 32.68 34.04 GA % Ery % 35.64 21.5 28.85 31.1430.37 25.44 GA % Xyl % 38.26 26.35 28.19 22.3 29.72 26.95 Man: Ery — 1.3— 0.85 — 1.33 Man: Xyl — 1.06 — 1.19 — 1.26 0.10% 2.50% GA % Man % 60.1526.65 48.24 38.51 50.56 29.31 GA % Ery % 56.47 18.21 43.35 6.83 49.2612.98 GA % Xyl % 56.94 17.61 44.8 15.29 46.29 22.82 Man: Ery — 1.46 —5.63 — 2.25 Man: Xyl — 1.51 — 2.51 — 1.28 0.30% 2.50% GA % Man % 71.5713.44 61.95 22.43 69.63 32.28 GA % Ery % 71.86 11.94 66.14 17.29 64.3612.64 GA % Xyl % 71.18 10.04 60.61 10.87 55.65 8.63 Man: Ery — 1.12 —1.29 — 2.55 Man: Xyl — 1.33 — 2.06 — 3.74

TABLE 7 Comparative data of % Protection and % Synergism of (18βGlycyrrhizin/Mannitol, Xylitol and Erythritol) ALT etc ALT etc MDA MDA %Prot. % Syn % Prot. % Syn 0.10% 1% GA % Man % 29.13 5.25 55.41 16.41 GA% Ery % 24.48 −5.83 46.38 14.01 GA % Xyl % 27.19 6.63 50.02 16.68 0.10%2.50% GA % Man % 40.35 10.52 85.62 28.23 GA % Ery % 40.06 −0.62 75.2916.58 GA % Xyl % 38.2 10.18 76.51 24.71 0.30% 2.50% GA % Man % 49.4−1.09 76.54 −21.21 GA % Ery % 52.68 −0.89 80.3 −15.44 GA % Xyl % 46.9−1.86 80.52 −12.22

TABLE 8 % Protection of Sucrose, Mannose, Xylose & Lactose GSH SOD etcTNF-α ALT etc MDA % Prot. % Prot. % Prot. % Prot. % Prot. Suc % 1   65.16 6.13 6.70 8.27 2.5 11.63 10.49 14.18 13.89 18.92 Mans % 1   6.123.93 7.85 6.14 10.65 2.5% 13.59 11.18 16.49 16.34 23.67 Xyls % 1   6.237.83 6.44 8.06 6.28 2.5 11.84 19.1 13.98 14.73 15.38 Lac % 1   4.36 6.788.19 8.21 7.70 2.5 14.8 17.38 15.26 17.41 21.47

TABLE 9 % Protection and % Synergism of (18β-GA: Sucrose, Mannose,Xylose & Lactose) Sample GSH GSH SOD etc SOD etc TNF-α TNF-α ALT etc ALTetc MDA MDA Code GA % % Prot. % Syn % Prot. % Syn % Prot. % Syn % Prot.% Syn % Prot. % Syn Suc % 10 0.1 1 10.65 14.64 18.32 10.37 15.14 9.9514.63 1.69 25.87 6.72 11 0.3 2.5 33.41 8.72 41.3 8.37 40.12 13.46 31.4−7.47 56.53 −13.39 Mans % 14 0.1 1 11.02 17.11 18.05 17.29 17.07 10.215.71 8.66 28.82 8.26 15 0.3 2.5 37.58 14.96 42.02 9.16 43.19 14.6533.88 −7.97 59.27 −15.35 Xyls % 18 0.1 1 10.9 14.05 20.97 8.83 15.6 10.816.84 4.26 22.23 −0.09 19 0.3 2.5 34.27 10.76 53.23 13.21 38.1 8.3632.28 −9.47 52.64 −14.66 Lac % 22 0.1 1 8.57 12.03 19.47 6.79 17.2 8.6516.75 3.17 25.1 6.04 23 0.3 2.5 38.16 12.57 47.19 5.07 39.55 8.53 34.6−9.98 57.88 −14.66

The data provided in the above tables clearly indicates that the18β-GA/D-Mannitol combination exhibits superior order of synergism overthe combination of 18β-GA/D-Erythritol and 18β-GA/Xylitol combinations.

The data provided in the above tables also indicates that overall the18β-GA/D-Mannitol combinations exhibit almost similar order of synergismas that of 18α-GA/D-Mannitol combinations.

Also it can be concluded that the combination of 18β-GA/reducing ornon-reducing mono or disaccharide has exhibited lesser degree ofsynergistic effect.

The present invention is illustrated with the following examples.However, it should be understood that the scope of the present inventionis not limited by the examples in any manner. It will be appreciated byany person skilled in this art that the present investigation includesfollowing examples and further can be modified and altered within thescope of the present invention.

Materials and Methods Reagents

Distilled ethanol was obtained from Bengal Chemicals, West Bengal,India. Biochemical kits like AST, ALT, ALKP and total protein wereobtained from Span Diagnostics Ltd. Surat, India. Time course study ofoxidative and nitrosative stress and antioxidant enzymes inK₂Cr₂O₇-induced nephrotoxicity. BMC Nephrol., 6: 4). TNF-α was estimatedby standard procedures as mentioned in Rat TNF-α ELISA kit (Bio Legend,Inc. San Diego, Calif., USA).

All the chemicals used in the present study were of analytical grade andobtained from the following companies: Sigma (St. Louis, Mo. USA), Merck(Mumbai, India), S. D. Fine Chemicals (Mumbai, India) and Qualign(Mumbai, India).

Alcohol Induced Sub-Acute Hepatotoxicity in Rats

Male Wistar albino rats weighing 150-200 g are procured from localregistered traders (CPCSEA Regd No. 1443/po/6/4/CPCSEA), Kolkata. Indiaand were acclimatized for 7 days at standard housing condition (26°C.±2° C., 60-70% RH with 12±1 hours light and dark cycle). Animals werefed with commercially available diet (Upton India Pvt. Ltd, India) andwater ad-libitum during the experiment period.

EXAMPLES Example 1 a) Model for Biological Testing

Male Wistar albino rats weighing 150-200 g are procured and randomlydivided into groups consisting of six animals in each group. Sub-acutetoxicity is induced by alcohol in rats by oral administration of 25%alcohol (2.5 gm/kg/day, p.o.) for 28 days and this group served as thenegative control and the positive control group received distilled wateronly.

b) Preparation of Drug Solution

All drug solutions were prepared in 15-40% aqueous alcohol, adjustingthe pH in the range of 4.0-9.0 for evaluation of hepato-protectiveactivity. This solution is further diluted with distilled water toobtain 25%, aqueous alcoholic solution and administered orally by gavageto different rats group of step (a).

c) Evaluation of Hepato-Protective Activity

On day 28^(th) day the animals are anaesthetized with ether and bloodsamples are collected by cardiac puncture and the serum is used for theassay of marker enzymes viz. serum alanine aminotransferase (ALT),aspartate aminotransferase (AST), alkaline phosphatase (ALP). The ratsare sacrificed by exposure to an overdose of ether, immediately afterthe collection of blood; their livers are removed, washed in coldsaline. Part of the liver is used for preparation of liver homogenate inphosphate buffer (pH 7.4). The supernatant is used for the estimation ofmalondialdehyde (MDA), super oxide dismutase (SOD), catalase (CAT),reduced glutathione (GSH), and Glutathione peroxidase (GPx).

Example 2

D-Mannitol (0.5 g) is dissolved in aqueous alcohol (100 ml) to provide0.5% solution. This solution is administered in several portions to oneof the rats group of Example (1a). The administration is carried outover a period of 28 days; each day 10 ml sample is diluted with 6 nildistilled water to make 25% aqueous alcoholic solution (16 ml) and fedorally (10 ml/kg/day). Evaluation of hepato-protective activity iscarried out as per Example (1c).

Mean % hepato-protection: ALT, AST and ALKP 12.26% SOD, CAT and GPx12.71% GSH 10.35% Hepatic MDA 19.17% TNF-α 7.19%

Example 3

D-Mannitol (2.5 g) is dissolved in aqueous alcohol (100 ml) to provide2.5% solution. This solution is administered in several portions to oneof the rats group of Example (1a). The administration, sample dilution,oral feeding and evaluation of hepato-protective activity is carried outas mentioned in Example 2 and as per Example (1c).

Mean % hepato-protection: ALT, AST and ALKP 29.71% SOD, CAT and GPx35.83% GSH 31.53% Hepatic MDA 50.80% TNF-α 31.46%

Example 4

18β-Glycyrrhizin (0.1 g) is dissolved in aqueous alcohol (100 ml) toprovide 0.1% solution. This solution is administered in several portionsto one of the of rats group of Example (1a). The administration, sampledilution, oral feeding and evaluation of hepato-protective activity iscarried out as mentioned in Example 2 and as per Example (1c).

Mean % hepato-protection ALT, AST and ALKP 8.38% SOD, CAT and GPx 11.45%GSH 3.29% Hepatic MDA 15.97% TNF-α 7.64%

Example 5

D-Mannitol (2.5 g) and 18β-Glycyrrhizin (0.1 g) are dissolved in aqueousalcohol (100 ml) to provide 2.6% solution. This solution is administeredin several portions to one of the rats group of Example (1a). Theadministration, sample dilution, oral feeding and evaluation ofhepato-protective activity is carried out as mentioned in Example 2 andas per Example (1c).

Mean % hepato-protection: ALT, AST and ALKP 40.35% SOD, CAT and GPx60.15% GSH 48.24% Hepatic MDA 85.62% TNF-α 50.56%

Example 6

D-Mannitol (2.5 g) and 18β-Glycyrrhizin (1.0 g) are dissolved in aqueousalcohol (100 ml) to provide 3.5% solution. This solution is administeredin several portions to one of the rats groups of Example 1(a). Theadministration, sample dilution, oral feeding and evaluation ofhepato-protective activity is carried out as mentioned in Example 2 andas per Example (1c).

Mean % hepato-protection: ALT, AST and ALKP 52.35% SOD, CAT and GPx78.75% GSH 83.29% Hepatic MDA 93.29% TNF-α 87.64%

Example 7

D-Mannitol (0.5 g) and 18β-Glycyrrhizin (0.1 g) are dissolved in aqueousalcohol (100 ml) to provide 0.6% solution. This solution is administeredin several portions to one of the rats group of Example (1a). Theadministration, sample dilution, oral feeding and evaluation ofhepato-protective activity is carried out as mentioned in Example 2 andas per Example (1c).

Mean % hepato-protection: ALT, AST and ALKP  21.0% SOD, CAT and GPx25.34% GSH 17.64% Hepatic MDA 39.63% TNF-α 19.16%

Example 8

D-Mannitol (3.0 g) and 18β-Glycyrrhizin (0.1 g) are dissolved in aqueousalcohol (100 ml) to provide 3.1% solution. This solution is administeredin several portions to one of the rats group of Example (1a). Theadministration, sample dilution, oral feeding and evaluation ofhepato-protective activity is carried out as mentioned in Example 2 andas per Example (1c).

Mean % hepato-protection: ALT, AST and ALKP 37.23% SOD, CAT and GPx58.15% GSH 45.53% Hepatic MDA 70.87% TNF-α 47.20%

Example 9

D-Mannitol (2.5 g) and 18β-Glycyrrhizin (0.4 g are dissolved in aqueousalcohol (100 ml) to provide 2.9% solution. This solution is administeredin several portions to one of the rats group of Example (1a). Theadministration, sample dilution, oral feeding and evaluation ofhepato-protective activity is carried out as mentioned in Example 2 andas per Example (1c).

Mean % hepato-protection: ALT, AST and ALKP 53.15% SOD, CAT and GPx79.83% GSH 76.38% Hepatic MDA 80.41% TNF-α 81.62%

Example 10

D-Mannitol/D-Xylitol/D-Erythritol (1.0 g) and 18β-Glycyrrhizin (0.1 g)are dissolved in aqueous alcohol (100 ml) to provide 1.1% solution. Thissolution is administered in several portions to one of the rats group ofExample (1a). The administration, sample dilution, oral feeding andevaluation of hepato-protective activity is carried out as mentioned inExample 2 and as per Example (1c).

Mean % hepato-protection: Sugar alcohols Enzymes/Markers D-MannitolD-Xylitol D-Erythritol ALT, AST and ALKP 29.13% 27.19% 24.48% SOD, CATand GPx 39.33% 38.26% 35.64% GSH 29.58% 28.19% 28.25% Hepatic MDA 55.41%50.02% 46.38% TNF-α 32.68% 29.72% 30.37%

Example 11

D-Mannitol/D-Xylitol/D-Erythritol (2.5 g) and 18β-Glycyrrhizin (0.3 g)are dissolved in aqueous alcohol (100 ml) to provide 2.8% solution. Thissolution is administered in several portions to one of the rats group ofExample (1a). The administration, sample dilution, oral feeding andevaluation of hepato protective activity is carried out as mentioned inExample 2 and as per Example (1c).

Mean % hepato-protection: Sugar alcohols Enzymes/Markers D-MannitolD-Xylitol D-Erythritol ALT, AST and ALKP 49.40% 46.90% 52.68% SOD, CATand GPx 71.57% 71.18% 71.86% GSH 61.95% 60.61% 66.14% Hepatic MDA 76.54%80.52% 80.30% TNF-α 69.63% 55.65% 64.36%

Example 12

DI-Mannose/D-Xylose/D-Lactose/D-Sucrose (2.5 g) and 18β-Glycyrrhizin(0.3 g) are dissolved in aqueous alcohol (100 ml) to provide 2.8%solution. This solution is administered in several portions to one ofthe rats group of Example (1a). The administration, sample dilution,oral feeding and evaluation of hepato-protective activity is carried outas mentioned in Example 2 and as per Example (1c).

Mean % hepato-protection: Sugars Enzymes/Markers D-Mannose D-XyloseD-Lactose D-Sucrose ALT, AST and ALKP 33.88% 32.28% 34.60% 31.40% SOD,CAT and GPx 42.02% 53.23% 47.19% 41.30% GSH 37.58% 34.27% 38.16% 33.41%Hepatic MDA 59.27% 52.64% 57.88% 56.53% TNF-α 43.19% 38.10% 39.55%40.12%

Example 13

D-Mannose/D-Xylose/D-lactose/D-Sucrose (1.0 g) and 18β-Glycyrrhizin (0.1g) are dissolved in aqueous alcohol (100 ml) to provide 1.1% solution.This solution is administered in several portions to one of the ratsgroup of Example (1a). The administration, sample dilution, oral feedingand evaluation of hepato-protective activity is carried out as mentionedin Example 2 and as per Example (1c).

Mean % hepato-protection: Sugars Enzymes/Markers D-Mannose D-XyloseD-Lactose D-Sucrose ALT, AST and ALKP 15.71 16.84% 16.75% 14.63% SOD,CAT and GPx 18.05 20.97% 19.47% 18.32% GSH 11.02 10.90% 8.57% 10.65%Hepatic MDA 28.82 22.23% 25.10% 25.87% TNF-α 17.07 15.60% 17.20% 15.14%

Example 14

D-Mannitol (1.0 g) and 18α-Glycyrrhizin (0.1 g) are dissolved in aqueousalcohol (I 00 ml) to provide 1.1% solution. This solution isadministered in several portions to one of the rats group of Example(1a). The administration, sample dilution, oral feeding and evaluationof hepato-protective activity is carried out as mentioned in Example 2and as per Example (1c).

Mean % hepato-protection: ALT, AST and ALKP 30.97% SOD, CAT and GPx42.42% GSH 32.74% Hepatic MDA 54.16% TNF-α 34.05%

Example 15

D-Mannitol (2.5 g) and 18α-Glycyrrhizin (0.3 g) are dissolved in aqueousalcohol (100 ml) to provide 2.8% solution. This solution is administeredin several portions to one of the rats group of Example (1a). Theadministration, sample dilution, oral feeding and evaluation ofhepato-protective activity is carried out as mentioned in Example 2 andas per Example (1c).

Mean % hepato-protection: ALT, AST and ALKP 46.49% SOD, CAT and GPx69.06% GSH 57.44% Hepatic MDA 78.80% TNF-α  68.1%

Example 16 Method of Preparation

0.1 to 0.4 grams of 18β/α-Glycyrrhizin is dissolved in 15-40% alcohol oralcohol:water mixture (in 100 ml). To this solution (0.5 to 3.0 grams)of sugar alcohol or sugar is added. The resulting solution is mixedthoroughly to obtain a clear solution. Thereafter the pH of theresulting solution is adjusted to between 4.0-9.0 and optionally desiredflavoring agent (vanilla) is added to obtain the final alcoholicbeverage composition.

The expansion for the abbreviations used in this application isenumerated as below:

-   -   GA: Glycyrrhizin (Glycyrrhizic acid or Glycyrrhizinic acid or        18β-Glycyrrhizin)    -   Man: Mannitol    -   Xyl: Xylitol    -   Ery: Erythitol    -   Mans: Mannose    -   Suc: Sucrose    -   Xyls: Xylose    -   Lac: Lactose    -   SOD etc: SOD, CAT & GPx    -   ALT etc: ALT, AST and ALKP    -   Mat: Matrine

Advantages of the Present Invention:

-   1. The alcoholic beverage of the present invention has better    hepato-protection.-   2. The alcoholic beverage of the present invention has an acceptable    odor, taste, clarity and acceptable buzz factor.

1. An alcoholic beverage composition providing synergistichepato-protection comprising of: a) hepato-protective agent(s) b)alcohol or water or a mixture thereof c) pH1 adjusting agent(s) and d)optionally flavoring agent(s).
 2. The beverage composition as claimed inclaim 1, wherein the hepato-protective agent is selected from a groupconsisting of 18β-Glycyrrhizin, 18α-Glycyrrhizin, sugar alcohols andsugars.
 3. The beverage composition as claimed in claim 2, wherein thesugar alcohol is selected from a group consisting of D-Mannitol,D-Xylitol, D-Erythritol and the sugar is selected from a groupconsisting of D-Sucrose, D-Mannose, D-Xylose and D-Lactose.
 4. Thebeverage composition as claimed in any of aforesaid claims, wherein thehepato-protective agent is a combination selected from group consistingof 18β-Glycyrrhizin, 18α-Glycyrrhizin and either one of D-Mannitol,D-Xylitol, D-Erythritol, D-Sucrose, D-Mannose, D-Xylose and D-Lactose ora mixture thereof.
 5. The beverage composition as claimed in claim 4,wherein 18β-Glycyrrhizin is in the range of 0.05 to 0.4%, preferably 0.1to 0.3% and D-Mannitol, D-Xylitol, D-Erythitol, D-Xylose, D-Mannose,D-Sucrose, D-Lactose or a mixture thereof is in the range of 0.5 to3.0%, preferably 1.0 to 2.5%; and wherein 18α-Glycyrrhizin is in therange of 0.05 to 0.3% preferably 0.1 to 0.3% and D-Mannitol, D-Xylitol,D-Erythitol or a mixture thereof, is in the range of 0.5 to 3.0%,preferably 1.0 to 2.5%.
 6. The beverage composition as claimed in any ofaforesaid claims, wherein more preferable hepato-protective agent(s) isa combination of 18β/18α-Glycyrrhizin and D-Mannitol.
 7. The beveragecomposition as claimed in claim 6, wherein 18β-Glycyrrhizin is in therange of 0.05 to 0.4%, and the D-Mannitol is in the range of 0.5 to 3.0%and preferably 18β-Glycyrrhizin in the range of 0.1 to 0.3% and theD-Mannitol is in the range of 1.0 to 2.5%; and wherein 18α-Glycyrrhizinis in the range of 0.1 to 0.3% and the D-Mannitol is in the range of 1.0to 2.5%
 8. The beverage composition as claimed in claim 1, wherein pHadjusting agent is an organic or inorganic base/buffer, preferablyselected from potassium sorbate or sodium phosphate (monobasic ordibasic or tribasic).
 9. The beverage composition as claimed in claim 1,wherein the flavoring agent(s) is selected from vanilla flavor,strawberry flavor.
 10. A process for the preparation of alcoholicbeverage composition of claim 1, comprising steps of (a) obtainingalcohol or water or a mixture thereof, (b) mixing 18β-Glycyrrhizin or18β-Glycyrrhizin with the alcohol or water or a mixture of alcohol andwater of step (a), (c) adding sugar alcohol or sugar to the mixture ofstep (b), (d) adjusting the pH of the resulting solution of step (c)between 4.0-9.0, (e) optionally adding the flavoring agent and (f)obtaining the required alcoholic beverage composition.
 11. Use ofalcoholic beverage composition as claimed in claim 1 for providingreduced hepato-toxicity.
 12. The alcoholic beverage as claimed in claim1 for use in a method of amelioration of diseases involving acute andchronic toxicity such as alcoholic liver diseases (ALD) like steatosis,steato-hepatitis, fibrosis, liver cirrhosis and hepatocellular carcinomaetc. caused by alcohol induced toxicity.